Aseptic Bioreactor Sampling System

ABSTRACT

We have modified a commercially-available adherent cell culture bioreactor, developing a new sampling manifold configuration and new way of taking samples to reduce contamination risk.

RELATED APPLICATIONS

This application is a Divisional of co-pending U.S. utility patentapplication Ser. No. 15/579,208 filed 3 Dec. 2017, which in turn assertspriority from Patent Cooperation Treaty application Serial No.PCT/US2017/025681 filed 3 Apr. 2017, which in turn asserts priority toUnited States provisional patent filing Ser. No. 62/322,651, filed 14Apr. 2016, the contents of which are here incorporated by reference.

GOVERNMENT INTEREST

None.

BACKGROUND

We have improved commercial-scale adherent cell culture by developing animproved bioreactor which provides a ˜50% increase in productivity vizprior art bioreactors, while also eliminating a source of contamination.

Commercially-available adherent cell culture bioreactors include, forexample, the laboratory-scale iCELLis™ Nano (commercially available fromPall Corporation, Cambridge Mass.) and the commercial scale iCELLis™ 500bioreactor, which provides a volume of up to 74 liters of cell culturemedium and a cell culture substrate of medical grade polyestermicrofibers which provide up to 500 m² growth area available to thecells. We improved the function of such commercial-scale bioreactors byfirst, defining the process steps in a small scale and then scalingthese up into a large scale. Pall Life Science, the manufacturer of theiCELLis™ brand of bioreactors, was recommending re-circulation orperfusion as a feeding strategy. The feeding strategy in small (Nano™)scale was tested by re-circulation, and later optimized by perfusion.

Perfusion is a process step where cells in a bioreactor are continuouslyfeed with a fresh medium at the same time removing equal amount of spendmedium, which enables the cell growth in high cell density (Vellinga etal. 2014). The perfusion rate can vary depending on the type of cellline used, the polypeptide product produced by those cells, the specificcell culture medium employed and the cell growth system used. Animportant aspect in the removal of the spent medium is also to removethe (possible toxic) metabolic side products from the cell culture.These side products may have a negative effect on cell viability, andfurther may impair the productivity of producer or host cells.

Options for perfusion include batch perfusion and fed-batch perfusion(where feeding of fresh medium is performed but no removal of spendmedium is performed). The fed-batch type approach is also are-circulation strategy, where the cell culture medium volume is onlyenlarged by external medium container and medium is re-circulated from amedia reservoir or container to the bioreactor and back again, whereinno actual removal of spend or used medium is done.

The manufacturer of the iCELLis™ line of bioreactors (Pall LifeTechnologies) markets the iCELLis™ 500 as suitable for perfusionfeeding. We made a very surprising finding during our first experimentwith iCELLis™ 500: as provided by the manufacturer, the equipment is notin fact capable of perfusion, if using the standard iCELLis™ 500 tubingand pump system set at the slowest pump output rate available.

We deconstructed the commercially-available apparatus and found out thereasons for failure. The commercially-available apparatus ismanufactured with the feed-out tube of larger interior diameter than thefeed-in tube. This configuration is intended to prevent unwantedoverflow of the liquid media by ensuring that in-flow cannot be greaterthan out-flow. We surprisingly found that this configuration adverselyimpacts pump efficiency and the productivity of the cultured cells.Rather, we found the feed-out tube is best of equal or smaller interiordiameter than the feed-in tube. We found that the commercially-provided(“stock”) medium feed-out tube was of too large interior diameter,providing a too-large interior volume space for the media flow rate.This large interior tube volume permitted formation of undesirable airpockets and bubbles in the tubing.

In addition, the stock feed-out tube was integrated into a too-weak pumpsystem which could not provide an adequate media flow out from thebioreactor. This may be because formation of air bubbles in turn appearsto affect the correct out feed-out pump capacity. The feed-out pumpworks by creating negative pressure in the feed-out tube, pulling mediainto the feed-out tube. Unlike liquid media, however, air bubbles expandin this negative-pressure environment, absorbing much of the negativepressure energy, thus frustrating pump work.

BRIEF DESCRIPTION

We resolved the failures inherent in the commercially-availableapparatus by retrofitting the bioreactor with a bigger peristaltic pumpable to provide the proper (lower) rate of fluid output, and replacingthe feed-out tubing with replacement tubing having a smaller insidediameter, thus preventing formation of undesirable air bubbles in thefeed-out tubing.

Another suggestion we have made for the manufacturer is to makebioreactors with smaller tubing diameter to improve the medium flowinside tubing.

We also have designed and tested a new sampling manifold configurationwhich eliminates the risk that a compromised aseptic filter willcontaminate an entire manufacturing batch.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a way to connect a bioreactor vessel to a samplingmanifold.

FIG. 2 shows a version of our improved sampling manifold.

DETAILED DESCRIPTION

We have implemented a way to improve medium feed in and feed outstrategy in large capacity adherent cell culture bioreactors such as,for example, the iCELLis™ 500 bioreactor. Commercially-available largescale bioreactors are designed to recirculate media at high velocity; wefound that perfusing media at a low velocity unexpectedly increasesproducer cell output. Our approach is the only suitable way to achieveacceptably slow and constant cell culture medium flow into and out ofthe bioreactor vessel. Constant medium flow supports high viralproductivity for infected, transfected or transduced producer cells,while avoiding excessive medium usage.

Adherent cell culture bioreactor vessels provide a substrate on whichcultured cells can adhere and grow. That substrate is housed in acontainer which contains liquid cell culture media.

Commercially-available large capacity adherent cell culture bioreactorsare provided with “Feed In” and “Feed Out” pumps. These pumpsrespectively pump cell culture media into and out of the container whichcontains the substrate, thus providing the cultured cells with freshculture media, and removing spent media and its appurtenant culturewaste products.

Example 1—Intermittent Pumping

Pumps can be programmed to run constantly. Run constantly at minimumspeed, the prior art iCELLis™ 500 Feed In and Feed Out pumps produce atleast about 42.3 L of medium flow per day (running at the slowest ratepossible, 24 rpm). The capacity of the bioreactor vessel being about 25liters, the medium in the reactor is completely exchanged almost twice aday.

We used a commercially-available large capacity adherent cell culturebioreactor vessel to culture transfected “producer” cells which expressviral polypeptides and produce viral particles. We found that theseproducer cells are most productive when the cell culture medium isperfused at a rate slower than the slowest pump output volume availablein commercially-available apparatus. To be able to get a properly lowmedium perfusion rate for our purposes, we programmed the pumps to runfor certain time interval, followed by an interval where the pumps didnot pump at all.

Because of this need to vary pump output, we have seen in practice thatthe stock iCELLis™ 500 bioreactor Feed Out pump provided by themanufacturer is not capable of removing medium out from the bioreactorvessel in a well controlled manner. We thus becan to test the viabilityof lower-output pumps able to provide an adequately low output flow.

Example 2—Constant Low-Velocity Pumping

We then investigated whether the variable media flow used in the priorart might impact cell culture in some way. To do this, we replaced theprior art Feed In pump provided with the iCELLis™ 500 bioreactor with areplacement pump which was able to provide a lower output volume, about16.7 L/day. For a 25 liter capacity bioreactor vessel, this means themedia in the vessel would be exchanged once every 3½ to 4½ days, ratherthan the nearly twice a day typical in the prior art. This reduces theflow across e.g., a 100 m² substrate surface area from at least 42.3 Lper 100 m² per day to 16.7 L per 100 m² per day. This lower Feed In pumprate enabled us to for the first time to run the Feed In pumpconstantly, without periodic stoppages.

During these runs it was observed that the Feed Out pump provided by themanufacturer was not able to perform the removal of the media from thebioreactor vessel throughout the process as planned. We thereforesimilarly replaced the prior art Feed Out pump provided by themanufacturer with a lower-output pump.

We performed a commercial-scale manufacturing run, using recombinantadherent producer cells to produce a recombinant adenovirus bearing atransgene (useful for e.g., gene therapy), using in the adherent cellculture process a combination of the stock iCELLis™ 500 Feed In pump andour own lower-output Feed Out pump. Our method is generally advantageousin producing vector with any kind of transgene (including therapeutictransgenes and marker transgenes such as green fluorescent protein), orany other genetic element or nucleotide sequence (e.g., viral vectorcontaining RNA transgene, shRNA, IngRNA, eRNA etc.).

We also performed three commercial-scale manufacturing runs, wherein weused our own lower-output Feed Out pump and also replaced the stockiCELLis™ 500 high-output Feed In pump also with a lower-output pump. Wereceived significantly higher adenoviral production in the adherentproducer cells in each of these three runs. In these three runs, theproductivity of viral particles per cell had increased 49.4% as comparedto productivity using the prior art higher-output pumps.

When we changed the process to work with lower-output pumps, our viralproductivity per cell surprisingly increased 49.4%. Without intendingfor the legal coverage of our patent to be bound by any scientificcausal theory, this improvement may be due to feeding the bioreactorvessel constantly with fresh medium, thereby keeping stable the level ofnutrients in the media in contact with the cultured cells. For example,we have found that adherent cells are most productive when the mediaflow is substantially constant and slow enough to maintain in the cellculture medium a level of lactate of not more than about 1.6 gramslactate/liter of culture medium. Similarly, we have found that certainadherent producer cells are most productive when the concentration ofglucose in the culture media is maintained at between about 0.5 andabout 1.0 grams of glucose per liter of media. Other adherent producercells are most productive with a glucose concentration which is higher(e.g., at least about 2.9 grams per liter) but nonetheless maintained ata relatively constant concentration due to low-velocity butsubstantially constant media flow. Alternatively, this may be due to theslow but constant flushing away of unwanted cellular waste products fromthe cell surface. Alternatively, this could be due to avoiding thephysical shear stress placed on the cultured cells when using thehigher-velocity media flow required by prior art hardware.Alternatively, this increase could be due to slower media flow enablingeach producer cell a longer time to produce virus-like particles.Whatever the cause (or causes), we found that a slower, constant mediumflow surprisingly and significantly increased adherent producer cellproductivity.

This increase is particularly surprising in light of the fact thatsuspension cell culture (e.g., the CultiBag RM™ suspension cell culturebag, commercially available from Sartorius Corp., Cambridge Mass.)provides more-or-less constant media flow across the cell surface, yetsuspension cell culture can be as less productive than adherent cellculture.

Lower-output pumps may also be successfully used for inoculation of thehost cells into the adherent bioreactor vessel, for drawing samples ofthe cell culture media during cell growth, and for harvesting e.g.,culture media at the end of the cell growth. We have surprisingly foundthat using a lower-output pump to perform these functionscounter-intuitively makes each of those processes faster than with theprior art high-output pumps.

One may replicate our system by simply obtaining acommercially-available large-scale adherent cell culture bioreactorvessel, disconnecting the Feed In and Feed Out tubing from the pumpsprovided with that bioreactor, and connecting that tubing to externalpumps. With external pumps, however, the operator may lose the abilityto control the external pumps through the computer which controls thebioreactor (and its integral pumps). For example, commercially availablelarge scale adherent bioreactors typically include a sensor whichmeasures the level of liquid cell culture medium in the bioreactorvessel, to monitor whether the cell culture substrate is adequatelysubmerged in medium. External pumps may not be automatically controlledby the feedback control loops that are automatically activated by theliquid cell culture medium level sensed in the bioreactor vessel.Similarly, commercially available large scale adherent bioreactorstypically include a recording device which records the running speeds ofthe pumps. Using external pumps, the output may not be recordedautomatically to the computer. One may overcome these disadvantages bycalibrating the external pumps before use and manually monitoring theculture medium flow in and out from the bioreactor vessel regularly, andby recording all steps manually to the batch manufacturing records.Alternatively, one can connect the new pumps to the bioreactor controlcomputer, so that the low output pumps can be controlled and monitoredsimilarly and simultaneously, as for the bioreactor itself.

The pump(s) can be controlled by software or through externalcalibration, or by controlling by nominal motor speed (RPMs), or using abalance or weight-controlled system. Alternatively, the pump(s) may becontrolled manually, or have an automatic speed control or any othersuitable way to control the pump. The precise control method is notcritical, as long as the pump is able to provide the consistent low-flowoutput we have surprisingly found is favorable to increased adherentcell production.

Our lower flow rate is effective for larger-area adherent cell cassettes(e.g., a 500 m² cassette). Lower flow rate may also be effectively usedwith smaller-area substrate cassettes, such as 66 m² fiber cassettes.

Our system can be used to culture adherent producer cells which expressviral polypeptide and thus produce recombinant virus or virus-likeparticles (we use the term “virus” in our appended legal claims toencompass both viruses and virus-like particles).

Example 3—Unidirectional Sampling Manifold

A bioreactor sampling manifold is used for taking liquid samples of thecell culture medium from the bioreactor vessel to e.g., check pH, or tomeasure glucose, metabolites and cell waste products (e.g., lactate) orother substances in the medium.

We unexpectedly found that the stock sampling manifold offered as partof the commercially-available iCELLis™ is vulnerable to contamination.We have successfully designed a new sampling manifold for the bioreactorfor removing the need of pumping medium in and out from the bioreactorvessel into a sampling bottle. This way we minimize the risk ofcontamination due to a sampling.

A schematic drawing of an adherent culture bioreactor is shown in FIG.2. The bioreactor is comprised of a vessel [I] which contains a cellculture substrate [2] to which cells adhere when cultured in adherentmode. The vessel [1] is filled with liquid cell culture medium whichirrigates the cell culture substrate [2]. The vessel [1] has a ventwhich includes an air filter [3] having an exit vent [4]. The exit vent[4] is able to vent excess gas out of the bioreactor vessel and thefilter [3] filters the gas flowing through the filter to preventparticulate contaminants (e.g., viral particles, fungal spores) fromexiting or entering the bioreactor vessel. The filter [3] is preferablydetachably connected to the vessel [1] via a vent tube [5].Alternatively, the filter may be integrated into the body of the vessel[1], albeit this risks enabling liquid cell culture media to splash ontothe filter and occlude it. Preferrably, the vent tube has a clamp [6]which enables a technician to close the vent tube [5] as and whendesired.

The vessel [1] also has a sampling tube [7] with an input end [7 a]which passes into the liquid cell culture medium and thus is able tointake liquid medium. Preferably, the sampling tube [7] also has a clamp[8] which enables a technician to close the sampling tube [7] as whendesired. The sampling tube [7] has a connection [9] enabling it to beconnected to a sampling device. The sampling device may be a detachablesampling bag or bottle. We prefer, however, the sampling device comprisea sampling manifold [FIG. 3] able to connect to a pair of detachablesampling bags or bottles.

The sampling manifold [FIG. 3] is connected [9] to the sampling tube[7]. The sampling manifold comprises a waste tube [10] having an inputend [10 a] and an output end [10 b], and the sampling tube [7] outputend [7 b] able to connect to a removable sampling container [11]. Thesampling container [11] may be, for example, a bag or a bottle.

The prior art commercially-available sampling manifold is based on theidea that first approximately 200 ml of cell culture media is merely arinse; it is pumped through the manifold to rinse the interior of themanifold. That rinse media passes out of the manifold through an exitopening into a temporary storage bottle. Additional cell culture mediais then pumped through the manifold, and a sample is collected. Afterthe sample is taken, the approximately 200 ml of cell culture rinsemedia which was initially collected in the temporary storage bottle ispumped from the bottle back into the manifold and back into thebioreactor vessel [1], so that as little as media as possible remainsinside the sampling manifold. The temporary storage bottle and thetubing can be emptied because the prior art configuration provides anaseptic filter in the sampling bottle, which filter permits ambient airto exit and enter the bottle (when liquid is pumped into and out of thebottle, respectively), the filter filtering airborne contaminants in theambient air and thus preventing contamination of the cell culturemedium.

If the integrity of the filter is compromised, however, this can pose amajor contamination risk which is difficult to detect until an entiremanufacturing batch has been contaminated. Using thecommercially-available prior art configuration, we have in fact incurreda contamination of a commercial-scale manufacturing batch. In evaluatingthe failure, it was observed that the aseptic air filter in a samplingbottle was broken, but it remained unclear how it had broken.

We thus designed a new sampling manifold using a different samplingtechnique. We have tested it successfully in at least fourcommercial-scale manufacturing batches, and have encountered noproblems.

With our sampling manifold we eliminate the need to pump cell culturemedia liquid out from the bioreactor vessel and then back again. Rather,with our sampling manifold, when liquid is taken from the bioreactorvessel, it is never pumped back in again. This reduces the risk ofcontamination significantly.

Our sampling manifold is described in FIG. 3. We prefer to make oursampling manifold from commercially-available C-flex tubing, Masterflex™tubing, clamps, and commercially-available polymer tube connectors. Thetubing can be non-sterile or sterilized by e.g., temperature- ormoisture-based techniques such as autoclaving, or gamma-irradiated orgas-based sterilization such as ethylene. The tubing can be ready-made,or custom-made by the user. We merely prefer it to be compatible withcommercially-available sterile connections, thus able to be branchedwith any type connections, and suitable for sealing and welding.

Our sampling manifold can be connected to a bioreactor such as theiCELLis™ 500 bioreactor through MPC-connector. When used with an iCELLisbioreactor, our sampling manifold can entirely replace the samplingmanifold offered by Pall Life Sciences as part of their commerciallyavailable Starter Kit, which is marketed together with the iCELLis™ 500bioreactor.

In our sampling manifold, we use a pair of tubes to take one sample. Onetube is used to rinse the manifold interior, the other tube is usedafter rinsing to take the sample. For example, one can use tube [15] torinse the manifold and then use tube [7] to take a sample.

To begin, a single-use sampling bag or bottle [11, 17, 19] isaseptically attached (we prefer through welding) to the sampling tube[7]. One may (and we prefer) to also attach a sampling bag [19] to thefirst (rinse) line of the pair [15]. Alternatively, the first line maydrain into a waste bottle etc.

To use our sampling manifold, liquid cell culture media is pumped fromthe bioreactor vessel [1] or an accompanying media reservoir through themanifold to rinse the interior. This is for rinsing the manifold andgetting fresh medium/liquid from the vessel [1] into the manifold. Thisrinse media is removed from the manifold via a rinse tube [15] into arinse media container [19]. Eherinse tube [15] is then sealed or closed.This may be done with a tubing clamp [12] (as illustrated).Alternatively, the tubing may be sealed with an end cap [on 15 b], orwelded closed [at 15 b], etc.: the manner of sealing is not particularlyimportant here. The rinse tube [15] thereafter remains sealed off forthe remainder of the manufacturing run. In the appended legal claims, werefer to sealing off for the remainder of the manufacturing run as“permanent” sealing because in the context of a given manufacturing run,it is effectively permanent (i.e., lasting to the end).

The sampling tube exit end [7 b], going to the sample container [11], isthen opened [13]. Fresh medium/liquid is pumped from the bioreactorvessel [1] (e.g., from the part of the bioreactor which houses theadherent cell culture substrate) into the sampling tube [7], and thenout of it [7 b] into the sampling container [11]. While we illustrateone sample container ([11]), one could alternatively use several samplecontainers and take several physically-separate samples. The samplingtube [7] is then closed [13]. The sampling container [11] may then besealed and removed from the sampling tube output [7 b]. After taking thesample, the pair of manifold branches [15, 7] remain sealed off for theremaining duration of the manufacturing run. We prefer both tubes bedrained to remove residual media, to reduce risk of contamination.

Sampling bags [11] provide a sampling port used for aseptically drawingliquid out of the sampling bag to assay. Several samples can thus betaken from one bag aseptically. To enable sequential sampling atdifferent times, we prefer to provide several pair of sampling tubes.With this, a second (or subsequent) sampling may be performed in asimilar fashion as the first one, using a different pair of manifoldtubes and a second sampling bag attached to the second tube of thatsecond pair of tubes. For example, to take a second sample, one may useline [10] as the rinse tube and line [16] as the sampling tube, with thesample being taken in container [17]). Media/liquid that has remained inthe manifold since the first sampling is discarded by pumping it out ofthe manifold [10 b], preferrably into a rinse or waste receptacle orcontainer until fresh media/liquid from the bioreactor substrate hasfilled the manifold. This second rinse tube [10] is then sealed orclosed. This may be done with a tubing clamp [12] (as illustrated).Alternatively, the tubing may be sealed with an end cap, or weldedclosed, etc.: the manner of sealing is not particularly important here.The second rinse tube [10] thereafter remains sealed off for theremainder of the manufacturing run.

The second sampling tube [16] is then opened [13]. Fresh medium/liquidis pumped from the bioreactor vessel [1] (e.g., from the part of thebioreactor which houses the adherent cell culture substrate) into thesecond sampling tube [16] and then out of it [16 b] into a secondsampling container(s) [17]. The sampling tube of the second pair [16] isthen closed [13]. The sampling container(s) [17] may then be sealed andremoved. After taking the sample, the pair of manifold branches [10, 16]remain sealed off for the remaining duration of the manufacturing run.

This configuration may be repeated as desired, providing as many pair ofrinse and sample tubes as desired to enable as many different samplingtimes as desired.

With our sampling port, a disadvantage at the moment is the need toconstruct the manifold (rather than be able to purchase itcommercially). Another disadvantage could be the use of several samplingbags for each manufacturing batch. These disadvantages, however, aremore than off-set by the reduced possibility of contaminating amanufacturing batch, which in commercial-scale manufacturing poses asignificant financial risk.

We claim:
 1. An adherent cell culture bioreactor comprising: a containerhaving an interior for containing liquid cell culture media and a solidsupport for adherent cell culture, the container interior in flowablecommunication with a first tube and a second tube, each of said tubescomprising an intake end in flowable communication with the containerinterior, and an output flush end and an output sample end, wherebyliquid cell culture media can flow from the container interior into theintake end of the tube, through the tube and exit the tube at the outputflush end or the output sample end, each tube able to be asepticallyclosed after cell culture media flows from the container interiorthrough the tube, each output sample end configured to be asepticallyattachable to a detachable sample container.
 2. The bioreactor of claim1, configured to contain at least about 20 liters of cell culture media.3. The bioreactor of claim 1, further comprising a thirdaseptically-closable tube comprising an intake end in flowablecommunication with the container interior, an output flush end, and anoutput sample end configured to be aseptically attachable to adetachable sampling container.
 4. The bioreactor of claim 2, the solidsupport for adherent cell culture providing at least about 60 m² ofsurface area for adherent cell culture.
 5. The bioreactor of claim 4,the solid support for adherent cell culture providing at least about 400m² of surface area for adherent cell culture.
 6. The bioreactor of claim4, where the solid support for adherent cell culture is fibrous.
 7. Thebioreactor of claim 1, further comprising liquid cell culture media andcells adherent to the solid support for adherent cell culture.
 8. Thebioreactor of claim 7, wherein the cells express a recombinantpolypeptide.
 9. The bioreactor of claim 8, where the recombinantpolypeptide comprises virus.
 10. The bioreactor of claim 9, where thevirus comprises a viral capsid containing a non-viral transgene.
 11. Amethod for aseptically sampling liquid media from a bioreactor,comprising: a. obtaining the bioreactor of claim 7, and then b. flowinga flush portion of the cell culture media from the container interiorinto the input end of the first tube, through the first tube and out theoutput flush end of the first tube, and then c. flowing a sample portionof the cell culture media from the bioreactor into the input end of saidfirst tube, through the first tube and out the output sample end of thefirst tube into a sample container, and then d. sealing said first tubewithout returning into the first tube the flush portion of cell culturemedia nor the sample portion of the cell culture media.
 12. The methodof claim 12, further comprising e. flowing a flush portion of the cellculture media from the bioreactor into the input end of the second tube,through the second tube and out the output flush end of the second tube,and then f. flowing a sample portion of the cell culture media from thebioreactor into the input end of the second tube, through the secondtube and out the output sample end of the second tube into a samplecontainer, and then g. sealing the second tube without returning intothe second tube the flush portion of cell culture media nor the sampleportion of the cell culture media.
 13. The method of claim 11, whereinthe bioreactor is configured to contain at least about 20 liters of cellculture media.
 14. The method of claim 13, the solid support foradherent cell culture providing at least about 60 m² of surface area foradherent cell culture.
 15. The method of claim 14, the solid support foradherent cell culture providing at least about 400 m² of surface area.16. The method of claim 11, wherein the cells express a recombinantpolypeptide.
 17. The method of claim 16, where the recombinantpolypeptide comprises virus.
 18. The method of claim 17, where the viruscomprises a viral capsid containing a non-viral transgene.
 19. Themethod of claim 14, where the solid support for adherent cell culture isfibrous.